Immunochemical determinations of antigens and antibodies

ABSTRACT

A NOVEL METHOD FOR THE IMMUNOCHEMICAL DETERMINATION OF ANTIGENS OR ANTIBODIES IN A LIQUID SAMPLE IN WHICH THEY ARE PRESENT IN LOW CONCENTRATION OR ARE ATTENDED WITH DISTURBING FACTORS, COMPRISING THE STEPS OF ADSORBING ONE OF THE IMMUNOCHEMICAL REACTION COMPONENTS ON A CARRIER, REACTING SAID COMPONENT WITH THE LIQUID TO BE TESTED, CONTAINING THE OTHER REACTION COMPONENT TO BE DETERMINED OR, IN CASE OF AN AGGLUTINATION IN HIBITION REACTION, REACTING SAID COMPONENT WITH A SOLUTION OF THE OTHER COMPONENT AND THE TEST LIQUID, SEPARATING THE CARRIER FROM THE REACTION MIXTURE, SUSPENDING THE CARRIER IN A SUITABLE LIQUID MEDIUM, AND READING THE SEDIMENT ATION PATTERN OF THE CARRIER.

United States Patent 3,565,987 IMMUNOCHEMICAL DETERMINATIONS OF ANTIGENS AND ANTIBODIES Antonius Hermanus Wilhelmus Maria Schuurs, Oss, Netherlands, assignor to Organon Inc., West Orange, N.J.,

a corporation of New Jersey No Drawing. Filed Oct. 20, 1967, Ser. No. 676,726 Claims priority, application Netherlands, Nov. 8, 1966, 6615722; June 16, 1967, 6708461 Int. Cl. G01n 31/06 US. Cl. 424--12 8 Claims ABSTRACT OF THE DISCLOSURE A novel method for the immunochemical determination of antigens or antibodies in a liquid sample in which they are present in low concentration or are attended with disturbing factors, comprising the steps of adsorbing one of the immunochemical reaction components on a carrier, reacting said component with the liquid to be tested, containing the other reaction component to be determined or, in case of an agglutination inhibition reaction, reacting said component with a solution of the other component and the test liquid, separating the carrier from the reaction mixture, suspending the carrier in a suitable liquid medium, and reading the sedimentation pattern of the carrier.

BACKGROUND OF THE INVENTION It is known that for the detection or estimation of antigens or antibodies the components required in the immunochemical reaction are combined in suitable volumes, after which the reaction takes place in the mixture of the components. Then the result is read from this mixture. Thus, for example, in the determination of the ABO blood group one drop of a suspension of erythrocytes is mixed in a tube or on a slide with one drop of the serum to be examined. After one hours incubation at room temperature the reaction is read.

In a similar manner the content of antibodies against thyroglobulin in serum of patients suffering from Hashimotos disease is determined with erythrocytes treated with tannin by the method of S. V. Boyden, described in J. Exp. Med. 93 (1951), 107, and then sensitized with thyroglobulin. One drop of diluted test serum is mixed on a slide with one drop of sensitized erythrocytes. The mixture is submitted to a rotating movement for 10 minutes and then the result is read.

L. Wide describes in Acta Endocrin. (1962), suppl. 70, the determination of human chorionic gonadotropin (HCG) by means of the haemagglutination inhibition test. Urine (0.4 ml.) is mixed in a round-bottomed tube with one drop of dilute antiserum and one drop of a suspension of erythrocytes. The tube is placed in a rack with a sloping mirror reflecting the bottom of the tube. During some 60 to 90 minutes the erythrocytes are allowed to settle; they then form a typical sedimentation pattern on the bottom of the tube which is read off.

Another example relates to the determination of the rheuma factor by the method of J. M. Singer and C. M. Plotz (Am. J. Med. 21 (1950) 888). 0.5 ml. of a suspension of polystyrene latex particles (diameter 0.8 micron) sensitized with human 'y-globulin is mixed with an equal volume of testserum in a dilute state, if desired. After incubation at 56 C. and then at 4 C. the tube with the reaction mixture is centrifuged for a certain time at a certain number of revolutions. Then the result is read from the turbidity of the supernatant liquid.

The last example is the detection or estimation of HCG in urine by means of latex particles to which the hormone is adsorbed as described in Netherlands patent ice application No. 6504823, filed Apr. 15, 1965. In this test system one drop of fluid to be tested, one drop of diluted antiserum to HCG, and one drop of a suspension of the sensitized latex are mixed on a glass slide. The result can be read after a few minutes of rocking the slide.

These methods produce usable results if the antigen or the antibodies to be determined are present in the test liquid in a suiiicient concentration and other components do not disturb the reaction. If either of these complications come into play, attempts are usually made to submit the liquid to be examined to concentration or fractionation.

An example thereof is the fractionation of sera of patients sulfering from rheumatoid arthritis to determine the rheuma factor. A survey of the various methods is given by K. J. Bloch (Bull. Rheum. Dis. 9 (1959) 185).

J. M. Dominguez and O. H. Pearson have described the determination of growth hormone in fractionated human sera by means of the haemagglutination inhibition reaction (cf. J. Clin. Endocr. Metab. 22 (1962) 865). The authors state that by the fractionation factors disturbing the reaction are separated from the hormone to be determined, the latter being concentrated simultaneously.

Another series of examples from the methods of urine concentration to determine the luteinizing hormone (LH).

Besides biological LH determinations in urine extracts immunochemical methods have been developed in recent years, e.g. by L. Wide (Acta Endocrin. (1962), suppl. and by D. R. Mishell (Am. J. Obst. Gyn. 95, 747 (1966) All methods adopted for the concentration of LH have certain disadvantages. One disadvantage is that during the treatment of the test liquid hormone may be lost. Another drawback of the methods is that any disturbing substances, too, may be concentrated together with the hormone. These defects render the accuracy of the results obtained doubtful.

SUMMARY OF THE INVENTION In accordance with the present invention, antigens or antibodies which are present in the liquid to be tested in low concentration, or are attended with disturbing factors can be detected and estimated immunochemically without prior concentration or fractionation by an agglutination or agglutination inhibition reaction, comprising the steps of adsorbing one of the immunochemical reaction components on a carrier, reacting said component or both components of the immunochemical reaction and the liquid to be tested, separating the carrier from the reaction mixture, suspending the carrier in a suitable liquid medium, and reading the sedimentation pattern of the carrier thus separated from the other components of the reaction mixture. The method of the invention can be used both in the immunochemical determinations based on an agglutination reaction between antigen and antibody and in those based on an agglutination inhibition reaction, in which the component of the test liquid to be determined can inhibit the agglutination.

The difference between the known procedures and that of the invention is that in the latter the carrier is separated from the other components of the reaction mixture prior to the reading of the result, while in the former the result is read from the carrier which is still in contact with the other components of the test liquid. The separation of the carrier from the rest of the reaction mixture provides the following advantages.

1) The exigencies of the reading of the test results imposes a limit to the maximal volume of the reaction mixture. For example, in the estimation of HCG by means of the haemagglutination inhibition reaction, described by Wide (vide supra), the volume of the reaction liquid is limited by the settling process of the erythrocytes, which process should be completed within a reasonable time. In a slide test the volume of the reaction mixture is limited to a few drops which can be held on a glass slide. However, in the present invention, the reaction between the various components is allowed to proceed in a larger volume than that feasible for the reading of the result. Thus, for example, in the determination of LH, which mostly occurs in urine in low concentrations only, 6 ml. of urine are mixed with 0.2 ml. of diluted antiserum to HCG, which cross-reacts immunochemically with LH, and 0.2 ml. of erythrocytes sensitized with HCG. The reaction between antigen and antibody takes place during an incubation following the mixing of the reagents. After that the erythrocytes are separated from the remaining part of the reaction mixture, preferably by centrifugation, then suspended in 0.5 ml. of a suitable buffer, if desired, and transferred to a tube with a perfectly regular, round bottom. Then the suspension is left to stand for 1 or 2 hours, after which the sedimentation pattern is read (cf. L. Wide, Acta Endocrin. (1962), supplement 70). While with the original method about 100 units HCG per litre can be detected, with the procedure according to the invention 10 units or even less than 10 units HCG per litre can be determined.

In the new method the contaminants of the test liquid, which can disturb the sedimentation pattern, are removed from the carrier particles prior to the reading of said pattern. The advantage of this procedure is illustrated by the following example. For the determination of HCG in serum of a pregnant woman 0.025 ml. of serum are mixed with 6 ml. of a suitable buffer. To this mixture are added 0.2 ml. of dilute antiserum and 0.3 ml. of a suspension of sensitized erythrocytes. After incubation of the reaction mixture the erythrocytes are isolated again and treated in a similar manner as described before.

The sensitivity of the method according to the invention can be raised by inserting in the procedure an incubation of test fluid and antiserum solution prior to the addition of the sensitized carrier particles. An incubation overnight at refrigerator temperature, or 2 hours at room temperature is appropriate. By this so-called pre-incubation the sensitivity can be raised by at least a factor two.

After the immunochemical reaction the carrier may be separated from the reaction mixture in any conventional manner, for example, by filtration, but centrifugation is preferred as this process allows rapid and complete separation. Preferably after having been washed, the resulting carrier may be observed directly, for example, after it has been transferred to a slide, to determine whether agglutination has taken place, or resuspended in a small volume of liquid and allowed to settle in a round-bottomed tube, after which the result of the reaction can be read from the sedimentation pattern.

For application of the invention use can be made of all kinds of known carriers, especially erythrocytes and latex particles.

The method is suitable for the demonstration of antibodies against various antigens, for example, antibodies against pathogenic bacteria which may be found in urine in low concentrations. It is also possible now to demonstrate antigens and antibodies occurring in serum in concentrations to that low degree that it has been impossible so far to demonstrate them in dilute sera, while dilution is necessary to prevent disturbance in agglutination.

Further it has been found that the method described above can be greatly simplified, especially when erythrocytes are applied as carriers, by using a test tube which may contain larger volumes of the substance to be examined and is proof against the forces occurring during centrifugation, and has moveover a perfectly regular, round bottom, and in which the particles may be resuspended. This enables all the stages of the process, including the sedimentation of the erythrocytes to an interpretable pattern, to be performed in the very same tube.

By shaping the tube so that it can be properly closed with a removable cap the components of the reaction mixture can be homogenized effectively by simply turning about. A test pack according to the invention comprises at least a test tube having a round transparent bottom, containing said antibodies or antigens or a mixture thereof, one of said components for the immunochemical determination being adsorbed on a carrier, and adapted to receive the liquid sample, to centrifuge the reaction mixture, and to observe the sedimentation pattern.

A further simplification is obtained by lyophilizing antiserum andsensitized erythrocytes in the said tubes in a suitable quanity andconcentration. Finally, antiserum and sensitized erythrocytes can be put together in a tube by lyophilizing them separately. In the latter case only the urine to be tested has to be put in the tube before performing the test.

When the tube contains lyophilized antiserum and/or erythrocytes it is essential that the removable cap referred to above should protect the contents from moisture to ensure a satisfactory stability.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the progress and the test pack in detail.

EXAMPLE 1 Determination of human chorionic gonadotropin (HCG) A phosphate buffer with pH 7 and having the following composition per litre is prepared:

Ethylenediamine tetra acetate (EDTA) 2 Nag-IP0 2 KH PO 0.72

NaCl 1.125

To this buffer is added 0.1% (wt./v0l.) bovine serum albumin. This buffer is used for the preparation of a series of dilutions of HCG. To 6 ml. of each HCG dilution are added 0.2 ml. of antiserum to HCG diluted to the desired strength with a citrate buffer of pH 6.5 containing 9 gm. of NaCl and 40 gm. of sodium citrate .2H O and 0.1% bovine serum albumin per litre, and 0.2 ml. of a 1.75% (v./v.) suspension of erythrocytes sensitized with HCG in the phosphate buffer. After incubating the mixture for 30 minutes at 37 C. it is centrifuged for 10 minutes at about 2,000 revolutions per minute. The erythrocytes are washed with 0.9% NaCl, after which they are taken up in phosphate buffer to obtain a total volume of 0.5 ml. The resulting suspension is transferred to a round-bottomed ampoule. After 2 hours the sedimentation patter is read. By this method 10 I.U. of HCG can be determined per litre.

EXAMPLE 2 Determination of human growth hormone (HGH) A series of dilutions is prepared of the growth hormone preparation to be examined in the bovine serum albumin containing phosphate buffer in accordance with Example 1.

To 6 ml. of each dilution are added 0.2 ml. of antiserum to HGH in citrate buffer in accordance with Example l, and 0.2 ml. of a suspension of erythrocytes sensitized with HGH (1.75% v./v.) in the phosphate buffer. The mixture is incubated for 30 minutes at 37 C., after which the erythrocytes are separated, washed with a physiological saline solution and taken up again in phosphate butter to obtain a total volume of 0.5 ml. Then the mixture is transferred to a round-bottomed ampoule. After 2 hours the sedimentation pattern is read.

The activity of the examined growth hormone preparation was 1.5 u. per mg. It appeared that HGH could still be demonstrated and determined in a concentration of 15 mu. or 10 ugm. per litre.

EXAMPLE 3 Determination of luteinizing hormone (LH) in urine from a woman with a normal menstrual cycle Of the urine to be examined the following series of dilutions is prepared: undilute, 1:2, 1:4, 1:8 and 1:16. The dilutions are prepared with a phosphate buffer in accordance with Example 1 containing 0.1% bovine serum albumin. Furthermore, a standard series is prepared of the Second International Reference Preparation for human menopausal gonadotripin (2nd IRP-HMG) in the same buffer in the concentrations 0, 12, 18, 25 and 35 International Units of LH per litre. Six ml. of test liquid with 0.2 ml. of a dilution of antiserum to HCG and 0.2 ml. of a suspension of 1.75% (v./v.) erythrocytes sensitized with HGG are incubated for 30' minutes at 37 C. The mixture is centrifuged and the erythrocytes are washed once with 0.0% NaCl and then suspended again in phosphate buffer. The suspension is transferred to a round-bottomed ampoule. After 2 hours the sedimentation pattern is read.

The LH-concentration of the urine is expressed in I.U. of LH per litre. LH can be determined in concentrations 25 I.U. of LH per litre. By this method the rise in the LH secretion about the time of the ovulation can be determined without concentrating the urine.

EXAMPLE 4 Determination of luteinizing hormone (LH) in urine from sterile patients The sensitivity of the estimation described in Example 3 can be raised by an incubation of the test fluid with antiserum solution prior to the addition of the suspension of sensitized erythrocytes. This so -called pre-incubation can be done overnight between 0 and C. The rest of the procedure is identical to that of Example 3. LH can be determined in concentrations greater than or equal to 10 LU. per litre taking the 2nd TRPHMG (see Example 3) as a standard. This method, with this level of sensitivity, can be used as a diagnostic tool to decide whether a case of sterility is of hypophyseal or of ovarian origin.

EXAMPLE 5 Determination of human serum albumin (HSA) A series of dilution is prepared of the HSA preparation, to be examined, in the phosphate buffer containing 0.1% bovine serum albumin in accordance with Example 1. To 4 ml. of each HSA dilution 0.2 ml. of a properly diluted rabbit anti-HSA serum is added. HSA and antiserum are allowed to react with each other during an incubation of hours at 5 C. To the reaction mixture is added 0.2 ml. of a suspension of sheep erythrocytes treated with formalin and tannin according to L. Wide, Acta Endocrin., Supplement 70 (1962) and sensitized with HSA. After the addition of these erythrocytes the procedure is as described in Example 1. The attainable sensitivity in this set-up is 2.5 g per litre. By raising the volume of the test fluid, e.g. from 4 ml. of 8 ml. the sensitivity is raised accordingly.

EXAMPLE 6 Determination of human chorionic gonadotropin (HCG) in serum To the serum from a sound woman are added 0, 1, 2, 3, and 6 LU. of HCG per ml. The serum is diluted 240x with phosphate buffer of Example. To 6 ml. of dilute serum are added 0.2 ml. of a solution of antiserum in citrate buffer (see Example 1) and 0.3 ml. of a suspension of 3.5% (v./v.) erythrocytes. After incubating the mixture for 30 minutes at 37 C. it is centrifuged, after which the erythrocytes are washed with phosphate buffer and finally taken up in the buffer to obtain a volume of 1.5 ml. Of this suspension 0.5 ml. is transferred to a round-bottomed ampoule. After 2 hours the reaction is read. By this method 3 I.U. HCG per ml. of serum can be detected.

EXAMPLE 7 Application of a special test tube The determination described in Examples 1 to 4 inclusive are performed in a tube made of glass or synthetic material, with a perfectly regular, round transparent bottom, an inner diameter of 11 mm. and a height of 10 or 11 cm., which is strong enough to stand centrifugal forces and has such a shape as to allow of its being closed easily with a removable cap, so that it is not necessary to transfer the washed and resuspended erythrocytes.

EXAMPLE 8 Application of a special test tube containing prelyophilized reagents Antiserum to purified luteinizing hormone prepared from human pituitaries diluted to the desired strength with the phosphate buffer of Example 1 is lyophilized in the test tubes described in Example 7 which are closed with a re movable cap in such a manner that no moisture can reach in the contents. The sensitized erythrocytes are lyophilized in a separate flask. Before the test the lyophilized erythrocycles are resuspended. The test liquid or a dilution thereof is mixed with lyophilized antiserum. To this mixture the desired quantity of sensitized erythrocytes is added, after which the test is performed further as described above.

In another test 0.5 ml. of a solution of antiserum, 0.5 ml. of phosphate buffer and 0.5 ml. of a suspension of erythrocytes are passed in succession into a test tube and frozen separately, in layers, in a mixture of solid carbon dioxide and acetone. Then the separate reagents are immediately lyophilized in the test tubes mentioned above by the method described in Netherlands Pat. No. 90530.

EXAMPLE 9 Determination of human chorionic gonadotropin (HCG) by means of a latex aggluatination inhibition reaction Bacto-latex of Difco Laboratories, Detroit, Mich., is sensitized with HCG as described in Example 1 of Netherlands patent application No. 6504823, filed Apr. 15, 1965. Two ml. of the phosphate buffer of Example 1 containing 0.1% bovine serum albumin, in which HCG has been dis solved in concentrations varying from 20 to 200 I.U. per

litre are mixed with 0.03 ml. (one drop) of a solution.

of antiserum in citrate butter (see Example 1) and 0.03 ml. (one drop) of a latex suspension. The reaction mixture is incubated for 30 minutes at 37 C. The latex is separated from the rest of the reaction mixture by centrifugation and suspended in the phosphate buffer with 0.1% bovine serum albumin to obtain an end volume of 0.1 ml. The resulting suspension is transferred to a slide and spread over a round surface, about 3 cm. in diameter. While rotating the slide slowly it is tilted a little from side to side. After 3 minutes it is ascertained whether agglutination has occurred. By this method quantities can be determined from 50 I.U. per litre.

What is claimed is:

1. Method for the immunochemical determination, by means of an agglutination reaction, of antigens or antibodies in an aqueous solution in which they are present in a concentration too low to be reliably determined by conventional agglutination reaction procedure without prior fractionation or concentration or are attended with factors disturbing to conventional agglutination reactions arising from contaminants present in the aqueous solution to be tested, comprising (a) adsorbing one of the immunochemical reaction components selected from the group consisting of antigens and antibodies on carrier particles suitable for antigens and antibodies;

(b) reacting said component by joining it with the aqueous solution to be tested containing the other reaction component to be determined;

(c) separating the carrier particles, after the reaction has taken place, from the reaction mixture;

(d) resuspending the particles in a small volume of a suitable aqueous medium; and

(e) visually determining the reaction pattern of the carrier particles.

2. The method of claim 1 in which the separated carrier particles are washed 'with an aqueous saline liquid not interfering with the immunochemical properties of the carrier particles and resuspended in an aqueous medium not interfering with the immunochemical properties of the carrier particles.

3. The method of claim 1, in which said carrier is separated by centrifugation.

4. The method of claim 1, in which said carrier consists of erythrocytes.

5. Method for the immunochemical determination, by means of an agglutination inhibition reaction, of antigens or antibodies in an aqueous solution in which they are present in a concentration too low to be reliably determined by conventional agglutination inhibition reaction procedure without prior fractionation or concentration or are attended with factors disturbing to conventional agglutination inhibition reactions arising from contaminants present in the aqueous solutions to be tested, comprising (a) adsorbing one of the immunochemical reaction components selected from the group consisting of antigens and antibodies on carrier particles suitable for antigens and antibodies;

(b) adding the other immunochemical reaction component to the aqueous solution to be tested;

(0) reacting the mixture obtained by (b) by joining it with the carrier particles obtained by (a);

'(d) separating the carrier particles, after the reaction has taken place, from the reaction mixture;

(e) resuspending the particles in a small volume of a suitable aqueous medium; and

(f) visually determining the reaction pattern of the carrier particles.

6. The method of claim 5, in which said carrier is separated by centrifugation.

7. The method of claim 5, in which said carrier consists of erythrocytes.

8. The method of claim 5, in which the separated carrier particles are washed with an aqueous saline liquid not interfering with the immunochemical properties of the carrier particles and resuspended in an aqueous medium not interfering with the immunochemical properties of the carrier particles.

References Cited OTHER REFERENCES Barron et al.: I. Immunology, vol. 90, pp. 908-813 (1963).

Singer et al.: Am. J. of Medicine, vol. 21, pp. 888-892 (December 1956).

ALBERT T. M-EYERS, Primary Examiner V. D. TURNER, Assistant Examiner 

